Delivery of disease control in aquaculture and agriculture using microbes containing bioactive proteins

ABSTRACT

A microbial biomass, made from algae, bacteria, fungi, yeast, or combinations thereof, provides a feed for animals raised either in agriculture or aquaculture. A feed additive, and a therapeutic composition can also be made from a microbial biomass of algae, bacteria, fungi, yeast, or combinations thereof. The feed, feed additive, and therapeutic composition can comprise one or more proteins, peptides, antibodies, antibody fragments, or a combination thereof, wherein said proteins, peptides, antibodies, antibody fragments, or a combination thereof are non-native to the microbes of the biomass. The biomass can have therapeutic, bioactive, nutritional, and/or immunogenic properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation-In-Part application ofPCT/US02/08651, filed Mar. 22, 2002, which claims the benefit of U.S.Provisional Application No. 60/277,947, filed Mar. 23, 2001, the entiredisclosures of which are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is directed to animal feeds used in aquaculture orin agriculture, with microbial cells as components. These microbialcells contain exogenous peptides, proteins, and/or antibodies, which canconvey resistance or immunity to pathogens (such as viral or bacterial),or otherwise improve the health and performance of the species thatconsume them. The microbial cells can be algae, bacteria, fungi, oryeast. The exogenous peptides, proteins, and/or antibodies can beexpressed inside the microbial cells by direct genetic modification ofthe microbe or by infecting the microbe with a virus that has beenaltered to express the protein of interest. The invention is alsodirected to animal feed supplements and therapeutics with microbialcells as components.

[0004] 2. Related Art

[0005] Plant products have been produced using specific geneticmodification to express proteins and/or antibodies of therapeutic value.The group at the Boyce Thompson Institute at Cornell has cloned a viralcoat protein into bananas capable delivering an oral vaccine wheningested by humans, but this concept has not been extended to microbes.

[0006] There are several plant biotech companies, such as Meristem,Large Scale Biology, and Prodigene, which are now expressing certainhuman therapeutic proteins, including antibodies, in plants. Large ScaleBiology is expressing proteins in tobacco plants using a tobacco mosaicvirus as a vector to produce the protein of interest. The protein isthen isolated and purified from the plant material and used for humantherapeutic purposes. In this way, the plant genome itself is actuallynot modified, but rather the genome of the infecting virus carries thegene of interest.

[0007] Recombinant microbes, including bacteria, yeast, and other fungi,have been used to produce human therapeutic proteins. However, suchrecombinant microbes have not been used in agriculture or agriculture,wherein the cultivated animal ingests the whole organism. Rather, todate, the recombinant organism has been used as a factory from which thetherapeutic protein is isolated and purified prior to use.

[0008] Certain plant products have been produced that contain proteinsand/or antibodies of therapeutic value. They have been produced byinfecting the plant with a virus that expresses the protein of interest.Large Scale Biology has a series of patents protecting this technology,but its purpose is to produce purified proteins for pharmaceuticalpurposes, which requires an extensive purification procedure followingharvesting of the plant material. These patents do not involve the useof the crude plant material as a source of both nutrition and diseasecontrol, except under the unusual condition that the pharmaceuticalproduct is expressed in the fruit of the plant.

[0009] Certain recombinant proteins have been produced in insect cellsusing an insect virus expression system (baculovirus). These proteinsare also produced in intact insect larvae following infection withmodified baculoviruses. In both cases, the insect cells or larvae areused as factories to produce the protein of interest, and therecombinant protein is then purified for pharmaceutical purposes. Insectcells or larvae infected with baculovirus are particularly useful in theexpression of certain human therapeutic proteins because thepost-translational modifications of the therapeutic proteins are similarto the post-translational modifications imparted upon expression inhuman cells.

[0010] A baculovirus expression system is an efficient method forexpressing proteins in insect cell culture. Baculovirus is in the familyBaculoviridae, a diverse group of large double stranded DNA viruses thatinfect arthropods, including insects, arachnids, and crustaceans.Baculoviruses are species-specific and do not infect vertebrates, norcan they propagate in mammalian cells in culture.

[0011] The Sindbis arbovirus can be used to deliver high levels of geneexpression in vivo in non-host arthropod species without causingcytopathic effects in infected cells or impairing the development of theorganism. A replication competent Sindbis virus containing the codingregion of green fluorescent protein (GFP) induced productive infectionswhen injected into insect larvae and pupae (Lewis, et al., 1999). Thus,virus-mediated ectopic gene expression has been accomplished inarthropods, a phylum that includes the classes Crustacea and Insecta.

[0012] Antibiotic doping is used routinely in the aquaculture setting.Typically, the pure or semipure antibiotics are added directly to thewater column. However, crude fermentation broths, or crude preparationsincluding cells, have not been used for any kind of therapeutic deliverysystem.

[0013] Production of amino acids, such as lysine, typically involves agenetically modified microorganism, which overproduces the amino acid ofinterest and excretes it into the fermentation medium. The wastestreamfrom such a fermentation would include biomass containing the aminoacid, and this wastestream product could be used as a crude deliveryform of the small molecule nutritive amino acid.

[0014] Microalgae (single celled algae or phytoplankton) represent thelargest, but most poorly understood, kingdom of microorganisms on theearth. As plants are to terrestrial animals, microalgae represent thenatural nutritional base and primary source of all the phytonutrients inthe aquatic food chain. As the primary producers in the aquatic foodchain, microalgae are the source of many phytonutrients, includingdocosahexaenoic acid (DHA) and arachidonic acid (ARA). Microalgae alsorepresent a vast genetic resource, comprising in excess of 80,000different species.

[0015] Yeast, filamentous fungi, and bacteria are also in the directfood chain of fish, crustaceans, and mollusks. However, only a very fewof these microbes, perhaps less than 10 species, have been exploited foraquaculture feeds. These few species have been used primarily forhistorical reasons and ease of cultivation. They have not been chosen onthe basis of any scientific evidence of superiority as nutritional ortherapeutic supplements.

[0016] The marine environment is filled with bacteria and viruses thatcan attack fish and shellfish, thereby devastating aquaculture farmsvery quickly. Bacteria and viruses can also attack single celledmicroalgae, so these organisms have evolved biochemical mechanisms todefend themselves from such attacks. Such mechanisms may involve thesecretion of probiotic compounds that inhibit bacterial growth or viralattachment.

SUMMARY OF THE INVENTION

[0017] The present invention provides a microbial biomass for use as afeed, feed additive, and/or therapeutic, and the use of such feed, feedadditive, and/or therapeutic to deliver a therapeutic dose of abioactive peptide or protein. The invention also provides a method forfeeding the feed, feed additive, and/or therapeutic to animalscultivated in agriculture and aquaculture.

[0018] This invention provides an aquacultural or an agricultural feedcontaining microbial biomass comprising one or more peptides, proteins,antibodies, antibody fragments, or a combination thereof, where theproteins and antibodies are non-native to the microbes of the biomass.Preferably, the microbes are selected from yeast or other fungi,bacteria, algae, or combinations thereof. The microbes can be engineeredto recombinantly express the proteins or antibodies recombinantly, orthe microbes can be infected with viruses or plasmids, which express therecombinant proteins or antibodies, e.g., without altering the genome ofthe host organism.

[0019] This invention similarly provides feed additives for animals andtherapeutic compositions for human and non-human animals. The biomasscan be extracted or purified to produce the therapeutic compounds.

[0020] This invention also provides a method of delivering therapeuticproteins to an animal comprising administering a feed comprising one ormore microbe expressing a non-native therapeutic protein to the animal.This method can be used to deliver therapeutic proteins to a non-humananimal subjected to intensive agricultural practices, or to fish orshellfish in aquaculture. The therapeutic microbes can be algae,bacteria, yeast, or filamentous fungi. The therapeutic protein can be arecombinant protein expressed by the microbe, e.g., the microbe can beinfected with a recombinant virus, which expresses a recombinanttherapeutic or bioactive protein. The method encompasses deliveringtherapeutic proteins that inhibit growth or replication of Vibriospecies in vitro, and proteins or peptides that inhibit Taura SyndromeVirus (TSV) or White Spot Syndrome Virus (WSSV) infection in shrimp. Italso encompasses recombinantly expressed antibodies, and fragmentsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1. Western blot of two Saccharomyces cerevisiae clones (A0244and A0245) containing Infectious Pancreatic Necrosis Virus (IPNV)sequence encoding segment A (containing genes for VP2, VP4, and VP3).Lanes 1, 2, and 15 show BioRad low molecular weight standards. Lanes 3and 4 show IPNV virus West Buxton strain proteins that areimmunoreactive with a polyclonal antibody to IPNV. Lanes 5, 7, 9, 11,and 13 show clone A0244 proteins; and lanes 6, 8, 10, 12, and 14 showclone A0245 proteins that are immunoreactive with the polyclonalantibody to IPNV. The clones in lanes 5 and 6 were harvested at 120hours post inoculation, the clones in lanes 7 and 8 at 96 hours, theclones in lanes 9 and 10 at 90 hours, the clones in lanes 11 and 12 at72 hours, and the clones in lanes 13 and 14 at 64 hours.

[0022]FIG. 2. Expression of White Spot Virus (WSV) genes in Escherichiacoli (BL21). His-tagged fusion proteins were detected with alkalinephosphatase with NBT/BCIP color development. Lane 1 is a negativecontrol (pET28 clone, no insert), Lane 2 illustrates VP35, Lane 3illustrates VP28, Lane 4 illustrates VP26, Lane 5 illustrates VP24, Lane6 illustrates VP19, and Lane 7 contains molecular weight markers.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Definitions

[0024] A “feed” is a preparation providing nutritional value to anyanimal, including, but not limited to, terrestrial animals, e.g.,humans, cattle, horses, pigs, sheep, goats, and poultry; and aquaticanimals, e.g., fish, shrimp, lobsters, crawfish, mollusks, sponges, andjellyfish.

[0025] A “feed additive” is any substance added to feed, regardless ofnutritional or therapeutic value.

[0026] A “therapeutic” is a substance that can heal, or provide aremedial, palliative, or preventive effect on a pathologic process.Therapeutics can be used to treat medical diseases, conditions, orsyndromes.

[0027] “Microalgae” include both prokaryotic and eukaryotic algae thatare classed in many different species. Prokaryotic algae are typicallyreferred to as cyanobacteria or bluegreen algae. Eukaryotic microalgaecome from many different genera, some of which overlap with themacroalgae, but can be generally differentiated by their size and lackof defined organs. Microalgae can have specialized cell types. Examplesof different groups containing microalgae include, but are not limitedto, the chlorophyta, rhodophyta, phaeophyta, dinophyta, euglenophyta,cyanophyta, prochlorophyta, and cryptophyta.

[0028] An “antibiotic” is a substance that can inhibit or stop thegrowth of microorganisms, or that can kill microorganisms.

[0029] “Bactericidal” refers to the ability to kill bacteria.“Bacteriostatic” refers to the ability to inhibit or stop the growth ofbacteria.

[0030] An “immunogenic epitope” is a discrete site of an antigenicmolecule against which an antibody will be produced, to which the T-cellreceptor responds, or to which an antibody can bind, and which iscapable of inducing an immune response.

[0031] “Passive immunity” is immunity conveyed by molecules, e.g.,antibodies, immunogens, other proteins, or sensitized lymphocytes, thatdeliver protection from antigens, and which is obtained from a sourceoutside the organism's own immune system. Passive immunity can beacquired by an oral route, e.g. from an organism, antibody, or othermolecule that enters the gastrointestinal system and provides immunity,e.g., by preventing infestation across the gastrointestinal mucosa, orby stimulating the gastrointestinal immune system, e.g., IgA antibodies,or gut-associated lymphoid tissue (GALT). Passive immunity can be alsoacquired by the transfer of antibodies from one animal to another, e.g.,the passive immunity an offspring acquires from its mother.

[0032] “Aquaculture” is the cultivation of aquatic organisms undercontrolled conditions. An “aquatic organism” is an organism grown inwater, either fresh- or saltwater. Aquatic organisms, include, but arenot limited to, fish, e.g., bass, striped bass, tilapia, catfish, seabream, rainbow trout, zebrafish, red drum, and carp; crustaceans, e.g.,penaeid shrimp, brine shrimp, freshwater shrimp, and Artemia; androtifers.

[0033] “Probiotic” refers to an organism or organisms important for thepromotion of the growth of another organism. Probiotic effects, e.g.,therapeutic or protective effects, can be delivered by probioticorganisms. Probiotic organisms include algae, bacteria, and fungi, suchas yeast. Herein the term probiotic activity includes the productionand/or secretion of a substance that defends the organism producing orsecreting the substance from bacteria or viruses that is sometimesreferred to as a prebiotic activity. For example, probiotics can inhibitbacterial and viral growth and attachment.

[0034] A “patient” is any living animal, including, but not limited to,a human who has, is susceptible to, or is suspected of having or beingsusceptible to, a pathologic condition, disease, or disorder, or whootherwise would be a subject of investigation relevant to a pathologiccondition, disease, or disorder. Accordingly, a patient can be an animalthat has been bred or engineered as a model for any pathologiccondition, disease, or disorder. Similarly, a patient can be an animal(such as a farm animal, a dairy animal, a ranch animal, an animal thatlives under water, an animal cultivated on land or in water for food orother commercial use, an experimental animal, or a pet animal) includinga human, who is serving as a healthy control for investigations intopathologic conditions, diseases, or disorders.

[0035] Embodiments of the Invention

[0036] Several algal species exhibit antibiotic activity. This activitycan be due to certain bioactive constituents in the membranes or cellwalls, the protein or the carbohydrate of the positively testing speciesthat inhibit bacterial growth (prebiotics or herein probiotics). Anystandard screening technique used to identify antibiotic agents can beused to screen for algae having antibiotic activity, includingincubating filter disks soaked in culture broth from the candidate algaeon a lawn of the target pathogenic microbe (e.g., Vibrio species). Thisinvention contemplates the use of these “friendly algae” in a probioticfashion to control the growth of certain pathogenic microorganisms in apond. This invention is further directed to the use of recombinantmicrobes or virus-infected microbes to deliver a bioactive protein ofchoice. The recombinant microbes or virus-infected microbes can betested for antibiotic activity by standard antibiotic screening assaysto confirm their activity.

[0037] Historically, only bacteria have been used in a probiotic fashionto alter a pond's ecology in order to eliminate or reduce the number ofpathogenic bacteria. A problem with the bacterial probiotic approach isthat the existing microbial ecology represents a massive buffer that isdifficult to modulate with the introduction of relatively small numbersof alternative bacteria, and the results to date have been unimpressive.Furthermore, even if the newly introduced bacteria do bloom, any largeincrease in bacterial levels in a pond can lower oxygen levels and causeharm to the other inhabitants, such as fish or shrimp.

[0038] Microalgae have not been considered before as probiotics.Previous experience in the screening of extensive algal culturecollections has indicated that a number of algal species exhibitantibacterial or bacteriostatic capabilities. Some of these activitiesmay include anti-Vibrio activity. Such species are candidates for a highvalue enrichment feed that delivers both nutritional and antibioticcapabilities. This invention provides an approach to disease controlthat provides a solution to an impending ecological disaster that willresult from the present uncontrolled practice of dumping toxic chemicalsand antibiotics into the water systems to control these bacterial,fungal, or viral pathogens.

[0039] Viral or bacteria infections can dramatically limit farmproductivity in terrestrial environments. The marine environment is alsofilled with bacteria and viruses that can attack fish and/or shellfish.Infection by bacteria or viruses can devastate intensive marine-basedfarms very quickly. One of the major disease control problems in shrimpaquaculture today is infection by certain viruses (e.g., White Spot,Taura, etc.). Conventional strategies, e.g., antibiotics, are noteffective in this situation, and shrimp cannot be vaccinated by methodsanalogous to those used for fish. Shrimp, like all crustaceans, haveonly a rudimentary immune system, so they are particularly susceptibleto devastation by viral attacks.

[0040] This invention provides a solution to this problem with abiological control method using a microbial biomass, e.g., microalgae asa vector to deliver anti-White Spot antibodies directly to shrimp. Such“designer feeds” can be a normal part of the diet, and can deliver atherapeutic dose of antibody directly to the shrimp's gastrointestinalsystem. This provides passive immunity; the exogenous antibody remainsoutside the host organism and prevents infestation through the gut wall.The invention envisions the use of transgenic algae, yeast, fungi and/orbacteria to deliver the antibody to the virus. Such probiotics, asenvisioned in the invention, do not have to replicate in the targetorganism for the desired effect to occur. Alternatively, the microbeitself may be infected with a virus that is engineered to produce theantibody of interest. Alternatively, the microbial source may deliver aportion of the virus (e.g. a coat protein or coat proteins) or fragmentthereof, in order to immunize the shrimp, other shellfish, finfish, orother aquatic or terrestrial animals.

[0041] Antibodies, or antibody fragments, to desired targets, such asWhite Spot Syndrome Virus or Taura Syndrome Virus, can be prepared byroutine immunization techniques, e.g., and selection of monoclonalantibody producing hybridomas, or by screening viral or bacterialexpression libraries of immunoglobulin genes and gene fragments. See“Current Protocols in Immunology,” Coligan, et al., eds, WileyInterscience, 1991, and periodic supplements. Nucleic acid sequencesencoding the binding sites of the selected antibodies can be clonedusing standard methods (see “Current Protocols in Molecular Biology,”Ausubel, et al., eds., Wiley-Interscience, 1987, and periodicsupplements), and antibodies can be expressed from recombinant microbes(including algae, see, e.g., U.S. Pat. No. 6,027,900) or cloned intoviruses that infect the desired microbes.

[0042] There are a number of well known bactericidal and bacteriostaticpeptides that inhibit microbial growth. These include, but are notlimited to, cecropins, penaeidins, bactenecins, callinectins, myticins,tachyplesins, clavanins, misgurins, pleurocidins, parasins, histones,acidic proteins, and lysozymes. These peptides can be made in a plantmaterial such as tobacco, soybean, corn, sunflower, cotton, safflower,canola, or any other agronomic species using recombinant methods wellknown to those in the art, and thus provided as a feed component toconvey resistance or tolerance to infestation. Suitable plant materialalso includes macroalgae (Kelps), which are grown worldwide as acommodity feed crop in aquaculture. Macroalgae are the foodstuffs ofmany aquaculture species, and this invention contemplates recombinantproduction of therapeutic proteins in the natural or farm diet ofjuvenile fish (e.g., half-grown catfish), as well as fish larvae. Thus,within the contemplation of this invention are macroalgae, or insects,or other host organisms that make up part of the food chain for thefeeding of larvae, juveniles, and adults in aquaculture, as well as thesame life sequence in the terrestrial animal feeds (e.g. pigs, chickens,and cows).

[0043] Post-harvest processing of some sort may be used to prepare thematerial for use as feeds. This invention contemplates conventional(known) processes for converting insect or plant material into feeds.Such conventional process includes homogenization followed by extrusioninto pellets of various sizes, depending on the application (e.g.,larval, juvenile, or adult). Other modes of preparation include spraydrying, fluid bed drying, or even providing the material as a liquidsuspension.

[0044] The invention provides a feed, feed additive, or therapeuticcomposition for an animal, which includes an algal biomass or any partsthereof, comprising one or more proteins, peptides, antibodies, antibodyfragments, or combination thereof, which are non-native to the biomass,and which can be chosen from eukaryotic algae or prokaryotic algaesources.

[0045] The algal biomass can comprise heterotrophic and/orphotosynthetic microalgae. The algae can be chosen from Synechocystis,and/or Chlorella strains. The algae can be probiotic.

[0046] The invention also provides proteins, peptides, antibodies,antibody fragments, or a combination(s) thereof which are expressedrecombinantly, e.g., by a recombinant virus.

[0047] The invention further provides embodiments in which the proteins,peptides, antibodies, antibody fragments, or combination thereof inhibitthe growth or replication of a pathogen, e.g., Vibrio, Taura SyndromeVirus, White Spot Syndrome Virus, and Infectious Pancreatic NecrosisVirus.

[0048] The invention further provides that the algal biomass, or anextract thereof, possesses antibiotic activity. The proteins, peptides,antibodies, antibody fragments, or a combination(s) thereof can bebactericidal and/or bacteriostatic. The protein, peptide, antibody,antibody fragment, or combination thereof can be, but is notnecessarily, chosen from cecropins, penaeidins, bactenecins,callinectins, myticins, tachyplesins, clavanins, misgurins,pleurocidins, parasins, histones, acidic proteins, and lysozymes.

[0049] The invention provides that the protein, peptide, antibody,antibody fragment, or a combination(s) thereof comprises an immunogenicepitope.

[0050] The invention provides a method of feeding an animal comprisingadministering to the animal a feed, feed additive, or therapeuticcomposition that includes a microbial biomass, such as an algal, fungal,e.g., yeast, or bacterial biomass, or any parts thereof, as well as oneor more proteins, peptides, antibodies, antibody fragments, orcombination thereof, which are non-native to the algal, fungal, e.g.,yeast, or bacterial biomass. The algal biomass can be chosen fromeukaryotic algae or prokaryotic algae sources.

[0051] The invention also provides that the proteins, peptides,antibodies, antibody fragments, or combination thereof can conferpassive immunity upon an animal. The animal can be raised inaquaculture, and can be a fish, e.g., a salmon, or a crustacean, e.g. ashrimp. Alternatively, the animal can be raised in agriculture, and canbe cattle, porcine, or fowl. The animal can also be a human.

[0052] The invention additionally provides the embodiments, as describedabove in relation to algae, for yeast or other fungi, and bacteria. Theyeast can comprise, e.g., a Saccharomyces strain. The fungi can comprisee.g., a Mortierella species. The bacteria can comprise e.g. aLactobacillus, Bacillus, or Bifidobacterium species.

[0053] Certain embodiments of the invention will now be described inmore detail through the following examples. The examples are intendedsolely to aid in more fully describing selected embodiments of theinvention and should not be considered to limit the scope of theinvention in any way.

EXAMPLE Example 1

[0054] Selection of Useful Microbial Sources for Feeds that ProvideDisease Control. Microalgal biomass samples, aqueous extracts, organicextracts, and extracts from the growth medium after cultivation of thealgae were concentrated and spotted on filter paper discs. Using steriletechniques, these discs were then placed on agar plates overlaid with alawn of selected test organisms including, but not limited to,gram-negative bacteria, gram-positive bacteria, antibiotic resistantbacteria, yeast, or other fungi. After incubation for an appropriatelength of time to allow growth of the lawn of test organism, the plateswere observed for zones of clearing (non-growth) around the filter paperdiscs. Large zones of clearing indicate potent antibiotic activity;small zones of clearing indicate less potent antibiotic activity.

Example 2

[0055] Incorporation of an Antibody into an Algal Feed. A particularviral or bacterial pathogen is chosen and used to prepare monoclonalantibodies using procedures well known to those in this field (Harlowand Lane, eds., 1988. Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press). Gene(s) coding for this antibody or anappropriate antibody fragment (F_(ab) or F_(v)) are isolated andamplified in the appropriate vector. The gene is spliced into atransformation vector suitable for a eukaryotic alga (e.g. Chlorella) ora prokaryotic alga (e.g. Synechocystis), or a yeast (e.g. Saccharomyces)or a fungus (e.g. Mortierella). The transformation vector is chosen sothat the antibody will be over-expressed in the microbial cell biomass.This biomass is then used as a feed additive in such a way as to providethe antibody directly to the animal, thus providing passive immunity.

Example 3

[0056] Expression of a Bactericidal Protein in a Microbial Feed. Abactericidal protein is chosen for the particular application. Forexample, proteins of the penaeidin class may be chosen for pathogeniccontrol in shrimp. Penaeidins are members of a family of antimicrobialpeptides isolated from crustaceans (e.g., Penaeid shrimp). Antimicrobialpeptides can also come from insects and chelicerates, and can include,but are not limited to, cecropins, peneaidins, bactenecins,callinectins, myticins, tachyplesins, clavanins, misgunins,pleurocidins, parasins, histones, acidic proteins, and lysozymes. Thegene for the chosen protein or peptide is either isolated from theoriginal source, an amplification source, or made synthetically. Thegene is then incorporated into a transformation vector suitable for aeukaryotic alga (e.g. Chlorella) or a prokaryotic alga (e.g.Synechocystis), or a yeast (e.g. Saccharomyces) or a fungus (e.g.Mortierella). The transformation vector is chosen so that the proteinwill be over-expressed in the microbial cell biomass. This biomass isthen used as a feed additive in such a way as to provide thebactericidal protein directly to the animal, thus providing resistanceto that particular pathogen.

Example 4

[0057] Vaccination Using Feeds. An antigen characteristic of aparticular pathogen is chosen as is indicated by the animal andcircumstances. For example, a viral coat protein(s) or componentthereof, or a protein from an infectious bacterium, or a componentthereof, is chosen. The gene coding for the protein(s) is isolated andincorporated into a vector suitable for use in the microorganism ofchoice. The transformation vector is chosen so that the protein(s) willbe over-expressed in the microbial cell biomass. This biomass is thenused as a feed additive in such a way as to provide the viral orbacterial or fungal protein(s) directly to the animal, thus stimulatingan immunological response to that particular pathogen. The microbialcomponent may enter the body of the animal in the digestive tract, orotherwise through contact in the air or water.

Example 5

[0058] Vaccination Using Probiotic Feeds. Probiotic bacteria such asLactobacillus, Bacillus, Bifidobacterium, etc. provide beneficialeffects by their presence as live organisms in the digestive tract of ananimal. As such, they are constantly replicating; they become asignificant portion of the intestinal microflora and make an excellentcontinuous delivery mechanism for oral vaccines. Oral vaccines deliverthe antigen to a portion of the intestinal mucosa where it can interactwith immunogenic tissues (e.g., Peyers Patches) and stimulate animmunogenic response.

[0059] An antigen characteristic to a particular pathogen is chosen asis indicated by the animal and circumstances. For example, a viral coatprotein or component thereof, or an infectious bacterial protein, or acomponent thereof is chosen. The gene coding for the protein is isolatedand incorporated into a vector suitable for use in the probioticmicroorganism of choice. Other gut microfloral components not generallyconsidered as probiotics, but which live in the intestine, such ascoliforms (e.g. Escherichia coli) can also be used as a vector forproducing the vaccine in situ.

[0060] The two viral coat proteins from salmon infectious pancreaticnecrosis virus (IPNV) are isolated and inserted into a transformationvector selected for use in Lactobacillus using molecular biology methodsthat are well known by those of skill in the art. The recombinantLactobacillus cells expressing the viral antigens as free proteins,excreted proteins, and/or virus like particles (assembled viruses withno nucleic acid) are then grown using conventional fermentationtechnology, harvested, and processed into a form usable as a salmonfeed. This form may include, but is not limited to, freeze drying, spraydrying, fluid bed drying, microencapsulation, extrusion, or tableting.The recombinant Lactobacillus is then provided to the salmon as a feed,thereby delivering both the valuable probiotic as well as the vaccine.In this case, the vaccine is constantly produced as long as therecombinant Lactobacillus is present in the gut of the animal.

Example 6

[0061] Delivery of Active Peptides or Proteins Using Probiotic Feeds.The gene for an active antimicrobial peptide, such as, but not limitedto, cecropins, peneaidins, bactenecins, callinectins, myticins,tachyplesins, clavanins, misgurins, pleurocidins, or parasins, or anantimicrobial protein (such as histones, acidic proteins, or lysozymes)is isolated and inserted into a transformation vector selected for usein Lactobacillus using molecular biology methods that are well known bythose of skill in the art. The recombinant Lactobacillus cells,expressing the free peptides or proteins or excreted proteins, are thengrown using conventional fermentation technology, harvested, andprocessed into a form usable as a feed for an animal such as, but notlimited to, fish, crustaceans, livestock, etc. This form may include,but is not limited to, freeze drying, spray drying, fluid bed drying,microencapsulation, extrusion, or tableting. The recombinantLactobacillus is then provided to the animal as a feed, therebydelivering both the valuable probiotic as well as the antimicrobialcompound. In this case, the antimicrobial compound is constantlyproduced as long as the recombinant Lactobacillus is present in the gutof the animal.

Example 7

[0062] Cloning and Expression of Structural Protein Genes of InfectiousPancreatic Necrosis Virus (IPNV) in Yeast. The West Buxton (WB) strainof IPNV (ATCC VR877) was purified as previously described (Yao andVakharia 1998). The virus was propagated in Chinook salmon embryo cellculture (CHSE-214; ATCC CRL-1681) at 15° C. in Eagle's minimum essentialmedium (EMEM) and supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL fungizone. Totalviral RNA was isolated from the purified virus by digestion withproteinase K (200 mg/mL) followed by a standard phenol:chloroformextraction (Sambrook et al. 1989).

[0063] Complementary DNA (cDNA) of a segment of IPNV encoding apolyprotein comprising the IPNV structural proteins was obtained byreverse-transcription polymerase chain reaction (RT-PCR), cloned intopCR2.1 and pUC18, and completely sequenced. Clones with 100% identity tothe published sequences for the VP2-NS-VP3 polyprotein were selected.These were removed from the cloning vector as an EcoRI fragment andligated into a pESC-URA yeast expression vector (Stratagene, LaJolla,Calif.) which was linearized with EcoRI and dephosphorylated with calfintestine alkaline phosphatase by standard methods (Sambrook et al.1989).

[0064] This expression vector was then used to transform XL1-B competentcells (Stratagene, LaJolla, Calif.) and colony selection was performedon Luria-Bertani agar with 12.5 μg/mL tetracycline and 50 μg/mLampicillin. Using blue color selection in the presence of X-gal andIPTG, only white colonies were selected. Plasmid preparations were madefrom selected colonies using the Qiagen QIAprep spin column method(Qiagen, Valencia, Calif.) as described by the manufacturer. Restrictiondigestion with EcoRI verified the plasmids were 6.6 kb, as expected ofpESC-URA.

[0065] Competent Saccharomyces cerevisiae YPH501 (Stratagene, LaJolla,Calif.) cells were made as described by the manufacturer. An overnightculture of YPH501 was diluted 1:20 in 50 mL of YPAD broth (1% yeastextract, 2% peptone, 0.0075% L-adenine hemisulfate, 2% dextrose) andgrown at 30° C. to A600 equal to 1.0. Cells were then pelleted at 1000 gfor 5 minutes at 4° C. The supernatant was discarded and the cellsresuspended in LTE buffer (0.1 M lithium acetate, 10 mM Tris-HCl (pH7.5), 1 mM EDTA). The resuspended cells were then centrifuged at 1000 gfor 5 minutes at 4° C. The supernatant was discarded and pelleted cellsresuspended in 0.5 mL of LTE buffer, and stored at 4° C. for one daybefore use.

[0066] Transformation of 50 μL of the YPH501 competent cells wasperformed in sterile 1.5 mL microfuge tubes by addition of 3 μg ofpurified IPNV/pESC-URA clone. The tube contents were mixed by inversionand incubated for 30 min at 30° C., and then heated to 42° C. for 15minutes in a water bath. The tube contents were plated at 100 and 200 μLper plate on Synthetic Dextrose Minimal Medium (SD dropout medium). SDdropout medium is auxotrophic, and composed of 6.7 g of yeast nitrogenbase without amino acids, 20 g dextrose, 1.3 g amino acid powder (13amino acids plus adenine sulfate, no added uracil), 20 g agar per literof medium. Plates were then incubated at 30° C. for 3 days, i.e., whencolony formation was evident. These colonies appeared as viable colonieson a background of yeast that was dead or not growing in the auxotrophicmedium, indicating they were capable of making their own uracil. Theseputative transformants were patch plated onto SD dropout plates andincubated again at 30° C. for 3 days. All of the clones selected wereable to grow on the auxotrophic medium. The structural genes VP2 and VP3have been reported to be expressed in a similar construct in insectlarvae (Vakharia, 2003), and to form virus-like particles.

[0067] Two transformed clones were then inoculated into SG dropoutmedium (SD dropout medium with the dextrose replaced by an equal amountof galactose) to induce the GAL10 promoter, which controls theexpression of the cloned foreign genes. Cells were grown at 30° C. withshaking for several days, then samples of the culture were periodicallyharvested and pelleted by centrifugation at 1500 g for 5 min at 4° C.Pelleted cells were broken by standard glass bead disruption techniques(Ausubel et al. 1997). The cells were resuspended in glass beaddisruption buffer (20 mM Tris-HCl (pH 7.9), 10 mM MgCl₂, 1 mM EDTA, 5%glycerol (w/v), 1 mM DTT, 0.3 M ammonium sulfate, 1 mM PMSF, 5 mMbenzamidine) and transferred to a 2 mL sterile Beadbeater tube (BiospecProducts, Bartelville, Okla.) that was pre-filled to ½ volume with 0.5mm acid-cleaned glass beads (Biospec Products). Cells were then placedon ice for at least 15 minutes. Cells were broken by pulsed-bead beatingwith 8 total pulses of 30 seconds with >1 minute intervals where thetubes were kept on ice. The Beadbeater tubes were centrifuged in amicrofuge for 5 minutes at maximum microfuge speed (14,000 rpm).Supernatants were transferred to clean 1.5 mL microfuge tubes; 50 μL wasremoved to a clean 10×100 mm glass tube for determination of theconcentration of total protein (Lowry et al., 1951) and the remainderstored at −20° C. until further processing.

[0068] Results of the Lowry assay were used to determine the volume ofsupernatant required to place 22 μg protein/lane into each of twopre-cast 12% polyacrylamide gels (BioRad, Hercules, Calif.) for sodiumdodecyl sulfate-electrophoresis (SDS-PAGE). The two gels were run at 150V (constant voltage) in the Micro Protean 3 system to separate thecomponent proteins of the supernatants according to their molecularweight. One gel was stained with Coomassie Blue to detect the componentproteins, and the proteins in the other gel were transferred directly toa nitrocellulose membrane by western transfer using the Micro Protean 3cell (Bio-Rad, Hercules, Calif.) as directed by the manufacturer.

[0069] Western blotting followed standard procedures (Ausubel et al.1997). Gels were equilibrated for 10-15 min in transfer buffer (25 mMTris, 190 mM glycine, 20% methanol) and assembled as described by theMicro Protean 3 manufacturer. The gels were transferred to thenitrocellulose membrane at 300 mA (constant current) for 2 hours, whilechilled with ice. The membrane was placed in TTBS buffer (20 mM Tris-HCl(pH 7.5), 0.1% Tween-20, 10 mM sodium chloride) and washed twice for 5min. The membrane was then blocked in blocking buffer (TTBS with 1%casein, 2% BSA) for one hour with shaking, then washed twice with TTBS.

[0070] The membrane was incubated at room temperature for 1 h withshaking with a sheep polyclonal antibody to IPNV (Microtek, Sannichton,British Columbia, Canada) at a 1:1000 dilution in TTBS with 0.05% BSA.Following this incubation in primary antibody, the membrane was washedtwice with TTBS then incubated with a secondary antibody, horseradishperoxidase-conjugated, affinity purified rabbit anti-sheep IgG (H+L)(Jackson Immuno Research, West Grove, Pa.), diluted 1:500 in TTBS with0.5% BSA and normal rabbit serum (Bioresource International, Camarillo,Calif.). The secondary mixture was incubated for a minimum of 1 h atroom temperature while shaking. The membrane was washed twice with TTBSthen 1 Step TMB Blotting was performed according to the manufacturer'sinstructions (Pierce Chemical, Rockford, Ill.). Upon sufficient colordevelopment, the reaction was stopped by rinsing the membrane withwater.

[0071] As seen in FIG. 1, multiple proteins are visible on the membraneas horizontal bands, distributed according to their molecular weight.The use of pre-stained molecular weight (MW) markers (Bio-Rad low MW) inlanes 1, 2, and 15 monitored the molecular weight distribution profileof the protein on the nitrocellolose post-transfer. The six major bandsin these lanes are 103, 77, 50, 34.3, 28.8, and 20.7 kDa, respectively,from top to bottom. Lanes 3 and 4 of the membrane contain the proteinsof the IPNV virus West Buxton strain, and the bands visible in FIG. 1demonstrate viral proteins that are immunoreactive with the IPNVantibody.

[0072] Yeast clones A0244 and A0245 express the same three majorimmunoreactive proteins of approximately 60, 32, and 29 kDa. Each of thethree proteins comigrated in the gel with an immunoreactive protein inthe virus. The 60 kDa protein corresponds to VP2 and the 32 kDa proteincorresponds to VP3.

[0073] As shown by the consistancy of the intensity of theimmunoreactive bands in lanes 5-14, expression of the recombinantprotein is maintained at a relatively constant level from the time theculture is started through the lag phase of growth, which indicates thatharvesting clones for maximal biomass will provide optimal levels ofrecombinant protein.

Example 8

[0074] Incorporation of IPNV Gene-Containing Clones A0244 and A0245 IntoFish Feed. The yeast mutants of Example 7 were grown in SG dropoutmedium at 30° C. with shaking for 5 days. Yeast were harvested bycentrifugation at 2300 rpm in a Jouvan B3.11 centrifuge for 15 minutesat room temperature. A gel-forming medium was produced by mixing 1.5%waxy maize digestible starch (Ulra-Sperce M, National Starch andChemical Co.), 1.2% sodium alginic acid (Sigma Chemical), and 4% AquaSavor (Bentoli), the volume adjusted to 120 mL of ddH₂O, and the mixturewarmed to dissolve the alginate (to about 40° C.). A stock solution of5% CaCl₂ (Sigma) and 1% NaCl (Sigma) was prepared with tap water in abeaker, filled approximately half full. The harvested yeast cells werecracked with glass beads using 4 mm glass beads (Biospec Products) and4×30 second pulses. The cracked cells were put into the gel-formingmixture at concentrations of 1% and 10%. The 1% yeast mixture wassupplemented with an additional 9% (weight/volume) yeast (Fleischmann'sDry Powdered Rapid Yeast). A control feed containing 10% non-recombinantyeast was also produced.

[0075] Feeds with control, 1% mutant, and 10% mutant yeast were producedby squirting the yeast mixture from 100 mL syringes into the CaCl₂/NaClsolution, which was gently mixing, such that upon contact, solidmaterials were formed, which were quickly mixed together into strands ofgelled feed. The gelled strands were strained through a screen withcourse gratings (1 mm) to provide materials of the correct size forfeeding small fish. The feed was then washed with tap water on a finescreen, and stored at 4° C. until fed to fish.

[0076] Hybrid striped bass (from 1-1.5 g each) were fed 1 g of one ofeither control, 1% mutant, or 10% mutant yeast at a rate of 0.5 g offood per day for a week, followed by a week of normal diet, followed byan additional week of test diet as a booster. Fish were housed in arecirculating system with 20 L tanks filled with Instant Ocean-basedartificial seawater. Plasma was collected from five fish at thebeginning of the study as controls. An additional five fish from eachtreatment were sampled at the end of the second study week (one weekafter stopping the initial exposure to the yeast). The remaining fishwere sampled at the end of the fourth week of the study, one week afterthe booster feeding. Blood was collected by caudal severing followingMS222 anesthesia; the caudal fin was removed with scissors, bloodcollected from the tail with a capillary tube, and centrifuged toisolate the plasma, which was stored until analysis.

Example 9

[0077] Cloning and Expression of White Spot Virus Genes in Bacteria.Five genes from shrimp White Spot Virus (WSV) were cloned from the DNAof WSV recovered from the hemolymph of WSV-infected shrimp using RT-PCRto amplify the genes and the TOPO TA cloning system from Invitrogen.Clones with VP35, VP28, VP26, VP24, and VP19 genes were compared to thepublished WSV gene sequences (van Hulten et al., 2001), and clones with100% identity selected for subcloning. The EcoRI fragments from the TOPOvector were sub-cloned into the pET28 vector from Novae (Madison, Wis.),previously cut with EcoRI and dephosphorylated. Clones containing EcoRIfragments were identified by color selection with IPTG and XGAL usingstandard methods (Sambrook et al. 1989). Protein expression wasdetermined by growing the clones in LB medium and inducing proteinexpression with IPTG. The pET28 vector tags expressed proteins with a6His tag, which was used to detect expression of the protein products ofthe WSV genes.

[0078] Expression was detected by SDS-PAGE followed by western blottingon Immobile-P membrane (Millipore) using standard methods (Sambrook etal. 1989). Anti-His antibody labeled with alkaline phosphatase coupledwith NBI/BCIP color development was used to detect expression of VP35,VP28, VP26, VP24, and VP19. As shown in FIG. 2, all five clones producedHis-tagged fusion proteins, which correspond to VP35, VP28, VP26, VP24,and VP19, respectively.

[0079] References

[0080] Ausubel F. et al. (1997) Short Protocols in Molecular Biology,3rd ed. John Wiley & Sons, Inc., New York.

[0081] Lowry O, Rosebrough N, Farr A, Randall R (1951) Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

[0082] Sambrook J, Fritsch E, Maniatis T (1989) Molecular Cloning: Alaboratory manual, 2 ed. Cold Spring Harbor Press, Cold Spring Harbor.

[0083] Vakharia V (2003) Sub-unit vaccine for infectious pancreaticnecrosis virus. In: U.S. Pat. Publ No. 2003/0,072,772 A1. UMBI, USA.

[0084] van Hulten M C et al. (2001) The white spot syndrome virus DNAgenome sequence. Virology 286:7-22.

[0085] Yao K, Vakharia V N (1998) Generation of infectious pancreaticnecrosis virus from cloned cDNA. J Virol 72:8913-8920.

1. A composition comprising (1) an algal biomass or any parts thereof,wherein the algal biomass is chosen from eukaryotic algae or prokaryoticalgae sources, and (2) one or more proteins, peptides, antibodies,antibody fragments, or combination thereof, which are non-native to thealgae of the biomass.
 2. The composition of claim 1, wherein thecomposition is a feed.
 3. The composition of claim 1, wherein thecomposition is a feed additive.
 4. The composition of claim 1, whereinthe composition is therapeutic.
 5. The composition of claim 1, whereinthe protein, peptide, antibody, antibody fragment, or combinationthereof is expressed recombinantly.
 6. The composition of claim 5,further comprising a recombinant virus that expresses the protein,peptide, antibody, antibody fragment, or combination thereof.
 7. Thecomposition of claim 1, wherein the composition inhibits the growth orreplication of a pathogen.
 8. The composition of claim 7, wherein thepathogen is Vibrio.
 9. The composition of claim 7, wherein the pathogenis chosen from Taura Syndrome Virus, White Spot Syndrome Virus, andInfectious Pancreatic Necrosis Virus.
 10. The composition of claim 1,wherein the protein, peptide, antibody, antibody fragment, orcombination thereof is bactericidal or bacteriostatic.
 11. Thecomposition of claim 10, wherein the protein, peptide, antibody,antibody fragment, or combination thereof is chosen from cecropins,penaeidins, bactenecins, callinectins, myticins, tachyplesins,clavanins, misgurins, pleurocidins, parasins, histones, acidic proteins,and lysozymes.
 12. The composition of claim 1, wherein the protein,peptide, antibody, antibody fragment, or combination thereof comprisesan immunogenic epitope.
 13. The composition of claim 1, wherein thealgal biomass comprises heterotrophic microalgae.
 14. The composition ofclaim 1, wherein the algal biomass comprises photosynthetic microalgae.15. The composition of claim 1 wherein the algae are chosen fromSynechocystis and Chlorella strains.
 16. The composition of claim 1,wherein the algae are probiotic.
 17. A method of feeding an animalcomprising administering to said animal a composition comprising (1) analgal biomass or any parts thereof, wherein the algal biomass is chosenfrom eukaryotic algae or prokaryotic algae sources, and (2) one or moreproteins, peptides, antibodies, antibody fragments, or combinationthereof, which are non-native to an organism of the algal biomass. 18.The method of claim 17, wherein the composition is a feed.
 19. Themethod of claim 17, wherein the composition is a feed additive.
 20. Themethod of claim 17, wherein the composition is therapeutic.
 21. Themethod of claim 17, wherein the protein, peptide, antibody, antibodyfragment, or combination thereof confers passive immunity upon theanimal.
 22. The method of claim 17, wherein the animal is raised inaquaculture.
 23. The method of claim 2, wherein the animal is chosenfrom a fish and a crustacean.
 24. The method of claim 23, wherein theanimal is chosen from a salmon and a shrimp.
 25. The method of claim 17,wherein the animal is raised in agriculture.
 26. The method of claim 25,wherein the animal is chosen from cattle, pigs, and fowl.
 27. The methodof claim 17, wherein the animal is a human.
 28. A composition comprising(1) a yeast biomass or any parts thereof, and (2) one or more proteins,peptides, antibodies, antibody fragments, or combination thereof whichare non-native to the yeast of the biomass.
 29. The composition of claim28, wherein the composition is a feed.
 30. The composition of claim 28,wherein the composition is a feed additive.
 31. The composition of claim28, wherein the composition is therapeutic.
 32. The composition of claim28, wherein the yeast biomass comprises a recombinant virus thatexpresses the protein, peptide, antibody, antibody fragment, orcombination thereof.
 33. The composition of claim 28, wherein thecomposition inhibits the growth or replication of a pathogen.
 34. Thecomposition of claim 33, wherein the pathogen is Vibrio.
 35. Thecomposition of claim 33, wherein the pathogen is chosen from TauraSyndrome Virus, White Spot Syndrome Virus, and Infectious PancreaticNecrosis Virus.
 36. The composition of claim 28, wherein the protein,peptide, antibody, antibody fragment, or combination thereof isbactericidal or bacteriostatic.
 37. The composition of claim 36, whereinthe protein, peptide, antibody, antibody fragment, or combinationthereof is chosen from cecropins, penaeidins, bactenecins, callinectins,myticins, tachyplesins, clavanins, misgurins, pleurocidins, parasins,histones, acidic proteins, and lysozymes.
 38. The composition of claim28, wherein the protein, peptide, antibody, antibody fragment, orcombination thereof comprises an immunogenic epitope.
 39. Thecomposition of claim 28, wherein the yeast is a Saccharomyces strain.40. The composition of claim 28, wherein the yeast are probiotic.
 41. Amethod of feeding an animal comprising administering to said animal acomposition comprising (1) a yeast biomass or any parts thereof, and (2)one or more proteins, peptides, antibodies, antibody fragments, orcombination thereof, which are non-native to the organism of the yeastbiomass.
 42. The method of claim 41, wherein the composition is a feed.43. The method of claim 41, wherein the composition is a feed additive.44. The method of claim 41, wherein the composition is therapeutic. 45.The method of claim 41, wherein the protein, peptide, antibody, antibodyfragment, or combination thereof confers passive immunity upon theanimal.
 46. The method of claim 41, wherein the animal is raised inaquaculture.
 47. The method of claim 46, wherein the animal is chosenfrom a fish and a crustacean.
 48. The method of claim 47, wherein theanimal is chosen from a salmon and a shrimp.
 49. The method of claim 41,wherein the animal is raised in agriculture.
 50. The method of claim 49,wherein the animal is chosen from cattle, pigs, and fowl.
 51. The methodof claim 41, wherein the animal is a human.
 52. A composition comprising(1) a fungal biomass or any parts thereof, and (2) one or more proteins,peptides, antibodies, antibody fragments, or combination thereof, whichare non-native to the fungi of the biomass.
 53. The composition of claim52, wherein the composition is a feed.
 54. The composition of claim 52,wherein the composition is a feed additive.
 55. The composition of claim52, wherein the composition is therapeutic.
 56. The composition of claim52, wherein the protein, peptide, antibody, antibody fragment, orcombination thereof is expressed recombinantly.
 57. The composition ofclaim 56, further comprising a recombinant virus that expresses theprotein, peptide, antibody, antibody fragment, or combination thereof.58. The composition of claim 52, wherein the protein, peptide, antibody,antibody fragment, or combination thereof inhibits the growth orreplication of a pathogen.
 59. The composition of claim 58, wherein thepathogen is chosen from Vibrio, Taura Syndrome Virus, White SpotSyndrome Virus, and Infectious Pancreatic Necrosis Virus.
 60. Thecomposition of claim 52, wherein the protein, peptide, antibody,antibody fragment, or combination thereof, is bactericidal orbacteriostatic.
 61. The composition of claim 60, wherein the protein,peptide, antibody, antibody fragment, or combination thereof, is chosenfrom cecropins, penaeidins, bactenecins, callinectins, myticins,tachyplesins, clavanins, misgurins, pleurocidins, parasins, histones,acidic proteins, and lysozymes.
 62. The composition of claim 52, whereinthe protein, peptide, antibody, antibody fragment, or combinationthereof comprises an immunogenic epitope.
 63. The composition of claim52, wherein the fungus is a Mortierella species.
 64. The composition ofclaim 52, wherein the fungal biomass or any parts thereof is probiotic.65. A method of feeding an animal comprising administering to saidanimal a composition comprising (1) a fungal biomass or any partsthereof, and (2) one or more proteins, peptides, antibodies, antibodyfragments, or combination thereof, which are non-native to the fungi ofthe biomass.
 66. The method of claim 65, wherein the composition is afeed.
 67. The method of claim 65, wherein the composition is a feedadditive.
 68. The method of claim 65, wherein the composition istherapeutic.
 69. The method of claim 65, wherein the protein, peptide,antibody, antibody fragment, or combination thereof confers passiveimmunity upon the animal.
 70. The method of claim 65, wherein the animalis raised in aquaculture.
 71. The method of claim 70, wherein the animalis chosen from a fish and a crustacean.
 72. The method of claim 71,wherein the animal is chosen from a salmon and a shrimp.
 73. The methodof claim 65, wherein the animal is raised in agriculture.
 74. The methodof claim 73, wherein the animal is chosen from cattle, pigs, and fowl.75. The method of claim 65, wherein the animal is a human.
 76. Acomposition comprising (1) a bacterial biomass or any parts thereof, (2)one or more proteins, peptides, antibodies, antibody fragments, orcombination thereof, which are non-native to the bacteria of thebiomass, and (3) a recombinant virus that expresses the protein,peptide, antibody, antibody fragment, or combination thereof.
 77. Thecomposition of claim 76, wherein the composition is a feed.
 78. Thecomposition of claim 76, wherein the composition is a feed additive. 79.The composition of claim 76, wherein the composition is therapeutic. 80.The composition of claim 76, wherein the protein, peptide, antibody,antibody fragment, or combination thereof, inhibits the growth orreplication of a pathogen.
 81. The composition of claim 80, wherein thepathogen is chosen from Vibrio, Taura Syndrome Virus, White SpotSyndrome Virus, and Infectious Pancreatic Necrosis Virus.
 82. Thecomposition of claim 76, wherein the protein, peptide, antibody,antibody fragment, or combination thereof is bactericidal orbacteriostatic.
 83. The composition of claim 82, wherein the protein,peptide, antibody, antibody fragment, or combination thereof is chosenfrom cecropins, penaeidins, bactenecins, callinectins, myticins,tachyplesins, clavanins, misgurins, pleurocidins, parasins, histones,acidic proteins, and lysozymes.
 84. The composition of claim 76, whereinthe protein, peptide, antibody, antibody fragment, or combinationthereof comprises an immunogenic epitope.
 85. The composition of claim52, wherein the fungal biomass or any parts thereof is probiotic.
 86. Amethod of feeding an animal comprising administering to said animal acomposition comprising (1) a bacterial biomass or any parts thereof, and(2) one or more proteins, peptides, antibodies, antibody fragments, orcombination thereof, which are non-native to an organism of thebacterial of the biomass, and (3) a recombinant virus that expresses theprotein, peptide, antibody, antibody fragment, or combination thereof.87. The method of claim 86, wherein the composition is a feed.
 88. Themethod of claim 86, wherein the composition is a feed additive.
 89. Themethod of claim 86, wherein the composition is therapeutic.
 90. Themethod of claim 86, wherein the polypeptide confers passive immunityupon the animal.
 91. The method of claim 86, wherein the animal israised in aquaculture.
 92. The method of claim 96, wherein the animal ischosen from a fish and a crustacean.
 93. The method of claim 92, whereinthe animal is chosen from a salmon and a shrimp.
 94. The method of claim86, wherein the animal is raised in agriculture.
 95. The method of claim94, wherein the animal is chosen from cattle, pigs, and fowl.
 96. Themethod of claim 86, wherein the animal is a human.
 97. The method ofclaim 86, wherein the composition expresses the protein, peptide,antibody, antibody fragment, or combination thereof.